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Toxicity to soil microorganisms

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Reference
Endpoint:
toxicity to soil microorganisms
Type of information:
experimental study
Adequacy of study:
key study
Study period:
15 June to 1 October 2015
Reliability:
1 (reliable without restriction)
Rationale for reliability incl. deficiencies:
other: Study according to international guideline (OECD guideline 216) under GLP. Validity criteria were met, deviations from protocol did not adversely affect study integrity.
Qualifier:
according to guideline
Guideline:
OECD Guideline 216 (Soil Microorganisms: Nitrogen Transformation Test)
Deviations:
no
GLP compliance:
yes (incl. QA statement)
Analytical monitoring:
not required
Vehicle:
yes
Details on preparation and application of test substrate:
AMENDMENT OF SOIL
- Type of organic substrate: Finely ground lucerne meal at 5.0 g/kg dw
- Other: deionised water to reah 45% of Water Holding Capacity

APPLICATION OF TEST SUBSTANCE TO SOIL
- Method: test concentrations were prepared from solutions in acetone. 8.0 g of Quartz sand was spiked with 2.0 ml of the proper solution of test substance in acetone and after complete evaporation the 8 g of quartzsand was mixed into 800 g (dw) of soil

VEHICLE:
- Chemical name of vehicle (organic solvent): acetone. The stock solution contained 406 mg test substance per ml acetone
- Concentration of vehicle in test medium (stock solution and final test solution): 2.0 ml of stock solution was used to spike 8.0 g of fine quartz sand
- Evaporation of vehicle before use: yes, passive evaporation unde s slow-running fume cupboard
Test organisms (inoculum):
soil
Total exposure duration:
28 d
Test temperature:
20 ± 2 °C
Moisture:
Max. water holding capacity (WHCmax) [g/100 g]: 35.4 ± 1.5
Soil moisture adjusted for test [% dry mass]: 15.9, equivalent to 45% of WHCmax
Initial soil moisture: 6.1% dry mass, equivalent to 17.2% of WHC
Details on test conditions:
TEST SYSTEM
- Testing facility: ECT Oekotoxikologie GmbH, Böttgerstr. 2 - 14, 65439 Flörsheim am Main, Germany
- Test container (type, material, size): Glass jars with lids (volume 580 mL, diameter 100 mm, Weck, Germany). The lids allowed for some gas exchange.
- Amount of soil: Each test vessel was loaded with 230 g soil fresh weight equivalent.
- No. of replicates per concentration: 4
- No. of replicates per control: 4
- No. of replicates per vehicle control: 4

SOURCE AND PROPERTIES OF SOIL
- Geographic location: uncultivated field at Offenbach, Rhineland-Palatine, Germany
- Supplied by: Landwirtschaftliche Untersuchungs- und Forschungsanstalt (LUFA), D-67346 Speyer, Germany
- Standard soil LUFA Type 2.3, a sandy loam
- Sampling location: Germany, Rhineland-Palatinate, Offenbach, field name „rechts der Landauer Str.“ field number 826/7.
- Sampling conditions: Depth: ca. 20 cm; Date: May 20, 2015; weather conditions: cloudy
- The sampling location of the soil was uncultivated during the last four years.
- Pesticide use history at the collection site: unclutivated and no pesticides applied during last four years
- Fertiliser: in 2014, 3500 kg CaO/ha was applied three times, in June, September, December and 1463 kg MgO/ha in December

- Preparation: Air drying (only until sievable) from May 20 to May 26, 2015; final sieving to 2 mm on June 01, 2015.
- Storage (condition, duration): The soil arrived at the test facility on June 05, 2015 and was stored at 8±2°C until June15, 2015, when it was acclimated at 22±2°C until the start of the test.
- Soil texture (if natural soil): sandy loam
- Particle size distribution: < 0.002 mm: 6.5 ± 1.6 %; 0.002 - 0.05 mm: 33.8 ± 0.1 %; 0.05 - 2.0 mm: 59.7 ± 1.6 %
- Soil taxonomic classification: LUFA SOil Type 2.3; Sandy loam
- Soil classification system: USDA
- Organic carbon (%): 0.67 ± 0.03 %
- Maximum water holding capacity (in % dry weigth): 35.4 ± 1.5
- CEC: 7.5 ± 0.9 mval/100 g
- pH (0.01 M CaCl2): 5.7 ± 0.6
- Initial nitrate concentration for nitrogen transformation test: 0.08 ± 0.01% (800 mg nitrate/kg dry weight):
- Initial microbial biomass as % of total organic C: 1.86 %
The microbial biomass carbon (Cmic) of the bulk soil (without lucerne meal added) was determined at the beginning of the test by applying the Fumigation-Extraction (FE) method according to ISO (1997). Carbon was extracted from three soil aliquots, each of non-fumigated soil and of fumigated soil. Cmic was estimated by the difference between the amounts of CaCl2-extractable carbon from fumigated and non-fumigated soil sample aliquots.
To meet the requirements of the test guideline OECD 216 the soil microbial biomass carbon content should be ≥ 1% of the soil organic carbon (Corg) content.

ADDITIONAL SUBSTRATE (N-source):
- Lucerne-green-meal pellets: purchased from Raiffeisen Waren-Zentrale Rhein-Main eG, Kraftfutterwerk Schierstein, Hafenweg 1, D-65201 Wiesbaden.
- Treatment: pellets were ground to a fine powder with an electric mill. The finely ground lucerne meal was incorporated in to the bulk test soil on day 0 of the test (test start) at a dosing of 5.0 g/kg dry weight soil immediately before test item application.
- Date of purchase: May 19, 2009
- Identification: Luzerne Gruenmehl Pellets
- C/N Analysis: LUFA Speyer, Germany
- Nitrogen content : 2.79 %
- Carbon content : 41.03 %
- C/N ratio: 14.7

EFFECT PARAMETERS MEASURED :
The concentration of nitrate was determined in aqueous extracts from soil aliquots on day 0, and day 28 after test item application.

VEHICLE CONTROL PERFORMED: yes

RANGE-FINDING STUDY
- Test concentrations: 0.1 - 1.0 - 10 - 100 - 1000 mg/kg soil dw
- Results used to determine the conditions for the definitive study: no effects up to and including 100 mg/kg. 77% reduction of nitrate concentrations in the 1000 mg/kg dose level.
Nominal and measured concentrations:
control, solvent control,
94.7 - 237 - 592 - 769 - 1000 mg/kg soil dw
Key result
Duration:
28 d
Dose descriptor:
EC10
Effect conc.:
217 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95%CL 90.83 - 313.92 mg/kg dw
Duration:
28 d
Dose descriptor:
other: EC20
Effect conc.:
318.96 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 955 CL 174.88 - 416.61
Key result
Duration:
28 d
Dose descriptor:
EC50
Effect conc.:
666.06 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Remarks on result:
other: 95% CL 551.77 - 794.52 mg/kg dw
Duration:
28 d
Dose descriptor:
NOEC
Effect conc.:
94.7 mg/kg soil dw
Nominal / measured:
nominal
Conc. based on:
test mat.
Basis for effect:
nitrate formation rate
Details on results:
- Effect concentrations exceeding solubility of substance in test medium: The test substance is highly insoluble but was applied using acetone as a carrier solvent.

The coefficient of variation (CV) of control samples regarding the nitrate concentration was 1.9% on day 0 and 1.3% on day 28 of the test. The CV of solvent control samples was 1.2% (day 0) and 3.6% (day 28). Since the maximum deviation was below 15%, the validity criterion was fulfilled.

Measured NO3concentrations of the NO3standard solution were 95.5% of nominal on day 0 and 95.9 % on day 28.

Nitrate concentration of the bulk soil used to set up the different treatments and controls was 86.9 ± 1.2 mg kg-1soil dw and was measured on day 0 before adding lucerne meal (mean and standard deviation, n = 4).

After adding lucerne meal on day 0 of the test, mean nitrate concentration of the control soil was 111 ± 2.2 mg kg-1soil dw and it increased to 117 ± 1.6 mg kg-1soil dw on day 28 (mean and standard deviation, n = 4). CV of the untreated control was 1.9% and 1.3% on day 0 and day 28, respectively.

Differences between the untreated control and the solvent control were statistically not significant; i.e. the solvent had no measurable effect on nitrate formation.

Nitrate concentration [mg/kg soil dw] in soils treated with test item on day 0 and day 28 and relative inhibition as compared to solvent control:

Test concentration

Day 0

Day 28

Inhibition [%]

[mg /kg soil dw]

mean ± sd

mean ± sd

 

0.0 (control)

111 ± 2.2

117 ± 1.6

 

0.0 (solvent control)

111 ± 1.3

118 ± 4.2

Not applicable

94.7

104 ± 4.7*

116 ± 2.7

1.3

237

105 ± 4.7*

99.6 ± 6.4*

15.5

592

99.3 ± 2.6*

70.7 ± 4.6*

40.0

769

107 ± 2.2*

52.2 ± 0.7*

55.7

1000

107 ± 4.0*

33.2 ± 0.9*

71.8

Mean ± standard deviation (sd) n = 4; * significantly different from the solvent control (Williams multiple sequential t-test, p<0.05, one-sided smaller).

On day 0, the nitrate concentration of test-item treated soils was significantly different from the nitrate concentration of the solvent control This significance, however, is not considered an effect due to the lack of any dose response relationship, i.e. no stronger effect at higher test concentrations.

On day 28, nitrate concentration in the soils treated with the test item at 237 mg/kg soil dw and above differed significantly from the nitrate concentration in the solvent control. Hence, the LOEC and NOEC were 237 and 94.7 mg/kg soil dry weight, respectively. At the level of the LOEC the inhibition was 15.5%.

Validity criteria fulfilled:
yes
Remarks:
Coeff of variation for nitrate concentration in controls and solvent controls was 1.9% and 1.2% on day 0 and 1.3 and 3.6% on day 28, respectively. Since this was below 15%, the validity criterion was fulfilled.
Conclusions:
The test item had a statistically significant effect on nitrate formation in soil at concentrations of ≥237 mg/kg soil dry weight. Thus the 28d-NOEC was 94.7 mg/kg soil dry weight, the 28d-EC50 was 666 mg/kg soil dry weight and the 28d-EC10 was 217 mg/kg soil dry weight.
Executive summary:

The effect of Linseed Oil, Oxidised, was studied in the Soil Microorganisms NitrogenTransformationTest according to OECD TG 216.

The test item was mixed via spiked quartz sand into sieved field soil amended with lucerne meal (5.0 g/kg soil dw), and incubated for 28 d at 20°C in the dark. Test solutions were prepared from a stock in acetone. They were used to soak defined amounts of quartz sand which were then mixed into portions of soil. A solvent control (SC) and an untreated control (C) and five test concentrations were tested with four replicates each. The test concentrations ranged from 94.7 mg /kg dw to 1000 mg /kg dw. Soil nitrate concentration was determined in soil extracts on day 0 and day 28.

The maximum coefficient of variation (CV) for nitrate of control samples was 1.9%. This is below the 15% required by the guideline, so the validity criterion was fulfilled.

On day 0, nitrate concentration of test-item treated soils was significantly different from the solvent control This significance, however, due to the lack of any dose response relationship, is not considered an effect. On day 28, nitrate concentration in the soils treated with the test item was significantly reduced compared to the solvent control at ≥237 mg/kg soil dw.

The dose-effect relationship observed on day 28 revealed a 50% effect concentration (EC50) of 666 mg/kg soil dw. The EC20 and EC10 were 319 and 217 mg/kg soil dw.

Description of key information

The 28d-NOEC was 94.7 mg/kg soil dry weight and the LOEC was 237 mg/kg dw. The 28d-EC50 was 666 mg/kg soil dry weight and the 28d-EC10 was 217 mg/kg soil dry weight.

Key value for chemical safety assessment

Long-term EC10 or NOEC for soil microorganisms:
217 mg/kg soil dw

Additional information

The toxicity of Linseed Oil, Oxidised, was studied in the Soil Microorganisms NitrogenTransformationTest (OECD TG 216) and in compliance with GLP. The test item was mixed via spiked quartz sand into sieved field soil amended with lucerne meal (5.0 g/kg soil dw), and incubated for 28 d at 20°C in the dark.Test solutions were prepared from a stock in acetone. The test concentrations ranged from 94.7 mg /kg dw to 1000 mg /kg dw.

After 28 days, the nitrate concentration in the soils treated with the test item was significantly reduced compared to the solvent control at ≥237 mg/kg soil dw. The EC50 was 666 mg/kg soil dw and the EC10 was 217 mg/kg soil dw.